In the developing retina, gradients of ephrin receptor (Eph) expression along the dorsal/ventral (EphB) and nasal/temporal (EphA) axes of the retina create positional information necessary for the topographic organization of retinal ganglion cell (RGC) axon terminals in the brain. Despite their importance in visual system development, the cellular mechanisms regulating the cellular and spatial patterns of Eph receptor expression in the retina are not well understood. The long term goal of this research program is to determine the transcriptional mechanisms that pattern the cellular and spatial patterns of EPH receptor expression in the developing retina. The central hypothesis of this proposal is that combinatorial effects of transcription factors and chromatin modifications regulate the nasal low/temporal high expression of EphA5 in retinal ganglion cells. We have cloned the EphA5 promoter and have data showing Pou4F2/Brn3b can activate the promoter in vitro. In the retina, Pou4 transcription factors are expressed specifically in RGCs and are required for their differentiation and the establishment of a normal retinotopic map. In Specific Aim 1, we will determine the role of transcription factors in regulating EphA5 promoter activity in RGCs. To test our working hypothesis that Pou4/Brn3 transcription factors regulate EphA5 expression RGCs, we will determine which Pou4 factors occupy predicted binding sites in the EphA5 promoter, if there are differences in promoter binding in the nasal and temporal retina and if loss of Pou4F2 disrupts EphA5 expression in the developing retina. We will then determine if Pou4F binding sites are necessary for EphA5 promoter activity in vitro and in vivo. Because Pou4 transcription factors are not expressed in gradients, additional mechanisms are required for down-regulating EphA5 expression in the nasal retina. We have identified spatial differences in methylation in EphA5 promoter methylation that correlate with spatial differences in mRNA expression and shown that methylation modulates EphA5 promoter activity. KLF transcription factors are expressed in RGCs in vivo and can differentially promote or block RGC axon outgrowth. In Specific Aim 2, we will determine the role of differential DNA methylation in regulating spatial differences in EphA5 expression in the retina. Our working hypothesis is that differential methylation regulates spatial patterns of EphA5 expression by altering recruitment of KLF transcription factors. To test this, we will determine the full extent of DNA methylation across the EphA5 promoter and identify which retinal cell types have differential methylation. We will determine which KLF transcription factors regulate the EphA5 promoter in vitro and how DNA methylation alters binding specificity and promoter activation. The proposed research will result in increased understanding of the mechanisms that regulate EphA5 expression in the retina. This information will be important for the future development of novel therapies to replace RGCs and regenerate the optic nerve, because successful restoration of functional vision will require new RGCs to re-establish topographically ordered connections in the visual centers in the brain. PUBLIC HEALTH RELEVANCE: Successful development of regenerative therapies to restore vision in patients with blinding diseases including glaucoma and optic neuropathy will require that regenerated retinal ganglion cells re-establish their connections to appropriate targets within the visual centers of the brain. This research seeks to understand the cellular mechanisms that control the normal expression patterns of specific genes that guide retinal ganglion cell axons and pattern their connections during development. This fundamental knowledge will be a critical component for identifying strategies to enhance optic nerve regeneration and, ultimately, to restore vision.